The long-term goal of this proposal is directed at the experimental determination of the mechanism(s) of protein folding using NMR methods. For these studies we incorporate specific fluorine labeled amino acids into proteins and use one-dimensional NMR to examine the rates of side chain stabilization during folding as well as the appearance and disappearance of folding intermediates. These rates are important because they may define rates of macromolecular interactions, ligand binding and posttranslational modifications in vivo. We plan to investigate, analyze and interpret the kinetics of side chain packing and stabilization during protein folding. Proteins currently under study include the E. coil dihydrofolate reductase, the intestinal fatty acid binding protein, PapD, a chaperone for pilus formation in pathogenic and bacteria and adenosine deaminase. Misfolded proteins have now been implicated in a number of neurological diseases, particularly Alzheimer's. In order to understand misfolding, we must understand the mechanism of folding itself. The lack of adenosine deaminase activity as a consequence of mutations distant from the active site, for example, leads to Severe Combined Immunodeficiency Disease in children. Experiments with wild type and mutant CuZu superoxide dismutase are also planned. Mutants of this enzyme are known to cause familial amyotrophic lateral sclerosis (fALS). A second project is to investigate proline isomerization during folding by directly measuring the rate of cis/trans isomerization of every proline in proteins containing multiple proline residues after incorporating 3-19F-proline. Both these projects employ novel real-time and equilibrium NMR methodology in addition to standard.